Journal: Canadian Respiratory Journal

Article Title: Polygonatum Polysaccharide Regulates Macrophage Polarization and Improves LPS-Induced Acute Lung Injury through TLR4-MAPK/NF- κ B Pathway

doi: 10.1155/2022/2686992

Figure Lengend Snippet: Effect of PSPs on the TLR4-MAPK/NF- κ B signaling pathway. Compared with control group, ∗ P < 0.05, ∗∗∗ P < 0.001; compared with LPS group, # P < 0.05, ### P < 0.001.

Article Snippet: The primary antibody included TLR4 (#14358, 1 : 2000, Cell Signaling Technology, USA), MAPK (p38, #9212, 1 : 2000, Cell Signaling Technology, USA), phosphorylated MAPK (p-p38, #4511, 1 : 1000, cell signaling technology, USA) and phosphorylated NF- κ B (p-NF- κ B, #3033, 1 : 1000, Cell Signaling Technology, USA), NF- κ B (#8242, 1 : 2000, Cell Signaling Technology, USA), CD206 (#24595, 1 : 1000, Cell Signaling Technology, USA), and β -actin (#3700, Cell Signaling Technology, USA).

Techniques:

Journal: Canadian Respiratory Journal

Article Title: Polygonatum Polysaccharide Regulates Macrophage Polarization and Improves LPS-Induced Acute Lung Injury through TLR4-MAPK/NF- κ B Pathway

doi: 10.1155/2022/2686992

Figure Lengend Snippet: Effect of PSPs treatment on NF- κ B nuclear translocation. Magnification: 10x, scale: 100 μ m.

Article Snippet: The primary antibody included TLR4 (#14358, 1 : 2000, Cell Signaling Technology, USA), MAPK (p38, #9212, 1 : 2000, Cell Signaling Technology, USA), phosphorylated MAPK (p-p38, #4511, 1 : 1000, cell signaling technology, USA) and phosphorylated NF- κ B (p-NF- κ B, #3033, 1 : 1000, Cell Signaling Technology, USA), NF- κ B (#8242, 1 : 2000, Cell Signaling Technology, USA), CD206 (#24595, 1 : 1000, Cell Signaling Technology, USA), and β -actin (#3700, Cell Signaling Technology, USA).

Techniques: Translocation Assay

Journal: Canadian Respiratory Journal

Article Title: Polygonatum Polysaccharide Regulates Macrophage Polarization and Improves LPS-Induced Acute Lung Injury through TLR4-MAPK/NF- κ B Pathway

doi: 10.1155/2022/2686992

Figure Lengend Snippet: Verification of PSPs impact on TLR4-MAPK/NF- κ B signaling pathway. Compared with control group, ∗∗ P < 0.01, ∗∗∗ P < 0.001; compared with LPS group, # P < 0.05, ## P < 0.01, and ### P < 0.001; compared with LPS + PSPs group, $ P < 0.05, $$ P < 0.01, and $$$ P < 0.001.

Article Snippet: The primary antibody included TLR4 (#14358, 1 : 2000, Cell Signaling Technology, USA), MAPK (p38, #9212, 1 : 2000, Cell Signaling Technology, USA), phosphorylated MAPK (p-p38, #4511, 1 : 1000, cell signaling technology, USA) and phosphorylated NF- κ B (p-NF- κ B, #3033, 1 : 1000, Cell Signaling Technology, USA), NF- κ B (#8242, 1 : 2000, Cell Signaling Technology, USA), CD206 (#24595, 1 : 1000, Cell Signaling Technology, USA), and β -actin (#3700, Cell Signaling Technology, USA).

Techniques:

Journal: Canadian Respiratory Journal

Article Title: Polygonatum Polysaccharide Regulates Macrophage Polarization and Improves LPS-Induced Acute Lung Injury through TLR4-MAPK/NF- κ B Pathway

doi: 10.1155/2022/2686992

Figure Lengend Snippet: Effects of PSPs on M1 and M2 phenotypic polarization and TLR4-MAPK/NF- κ B signaling pathway of mouse macrophages. (a) The effect of PSPs on M1 and M2 phenotypic polarization of mouse macrophages was measured by flow cytometry. (b) The expression of TLR4-MAPK/NF- κ B signal proteins in lung tissue. Compared with the control group, ∗∗ P < 0.01; compared with LPS group, ## P < 0.01, ### P < 0.001.

Article Snippet: The primary antibody included TLR4 (#14358, 1 : 2000, Cell Signaling Technology, USA), MAPK (p38, #9212, 1 : 2000, Cell Signaling Technology, USA), phosphorylated MAPK (p-p38, #4511, 1 : 1000, cell signaling technology, USA) and phosphorylated NF- κ B (p-NF- κ B, #3033, 1 : 1000, Cell Signaling Technology, USA), NF- κ B (#8242, 1 : 2000, Cell Signaling Technology, USA), CD206 (#24595, 1 : 1000, Cell Signaling Technology, USA), and β -actin (#3700, Cell Signaling Technology, USA).

Techniques: Flow Cytometry, Expressing

Journal: Journal of Skin Cancer

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Subversion of the Anti-Inflammatory Response in Human Skin Cells Reveals Correlates of Latency and Disease Pathogenesis

doi: 10.1155/2014/246076

Figure Lengend Snippet: Early activation of NF- κ B signaling during acute infection with rKSHV.219 is sustained in MeWo cells, but subsequently limited in Mel1700 cells following long-term infection. (a) MeWo and Mel1700 cells either mock-infected, treated for 20 min. with TNF- α , or infected with rKSHV.219 for 20 and 60 min. were used in western blot analysis for phosphorylated NF- κ B p65. Mock-infected cell lysates were used to determine baseline levels of phosphorylated NF- κ B p65, whereas lysates from cells treated with TNF- α served as positive controls for activation of NF- κ B p65. Total NF- κ B protein was used as an internal loading control. (b) Total RNA from half of the same cells used in (a) was used for RT-PCR amplification of the NF- κ B-controlled ICAM-1 gene. (c) MeWo cells (top) and Mel1700 cells (bottom) either mock-infected (control), TNF- α -treated, or infected with rKSHV.219 for 0.3 h, 1 h, 3 h, or 6 h were used in western blot analysis for phosphorylated NF- κ B p65. (d) Western blot analysis of the phosphorylation levels of key components of the NF- κ B-signaling pathway in long-term cultures of uninfected (−) and infected (+) MeWo and Mel1700 cells.

Article Snippet: Unless otherwise indicated, all rabbit monoclonal antibodies against key components of the NF- κ B pathway, as well as a polyclonal rabbit antibody to phosphorylated NF- κ B p65, were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction, Amplification

Journal: Journal of Skin Cancer

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Subversion of the Anti-Inflammatory Response in Human Skin Cells Reveals Correlates of Latency and Disease Pathogenesis

doi: 10.1155/2014/246076

Figure Lengend Snippet: A link between KSHV latency and the MC1-R signaling axis in skin-derived cell lines. (a) Western blot analysis of phosphorylated NF- κ B p65, MC1-R, and TRP-1 in total cell lysates extracted from MeWo (a) or Mel1700 (b) cells either uninfected (control) or acutely infected with KSHV for 0.3 h, 1 h, 3 h, or 6 h. GAPDH was used as loading control. (c) ImageJ quantitation of the p65 band intensities in (a) and (b) relative to GAPDH controls. (d) Mel1700 cells were infected in 6-well plates with increasing volumes (mL/well) of concentrated supernatant containing infectious KSHV, and total RNA from infected cells was subjected to RT-PCR using primer sets for host anti-inflammatory MC1R, POMC, and SLC7A11. (e) Equal aliquots from the same RNA used in (d) were subjected to RT-PCR analysis for select viral latency and cell growth control genes (i.e., GPCR, LANA, and v-FLIP). (f) Western blot analysis of the melanoma cell marker, Melan A, and anti-inflammatory genes MC1-R, TRP1, and SLC7A11 in total cell lysates of uninfected (−) or chronically infected (+) long-term cultures of MeWo-KSHV and Mel1700-KSHV cells. GAPDH was used as an internal control for both the RT-PCR (e) and western blot assays.

Article Snippet: Unless otherwise indicated, all rabbit monoclonal antibodies against key components of the NF- κ B pathway, as well as a polyclonal rabbit antibody to phosphorylated NF- κ B p65, were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Derivative Assay, Western Blot, Infection, Quantitation Assay, Reverse Transcription Polymerase Chain Reaction, Marker

Journal: Journal of Skin Cancer

Article Title: Kaposi's Sarcoma-Associated Herpesvirus Subversion of the Anti-Inflammatory Response in Human Skin Cells Reveals Correlates of Latency and Disease Pathogenesis

doi: 10.1155/2014/246076

Figure Lengend Snippet: Hypothetical model of immunophysiologic mechanisms that control transmission of KSHV from latently infected cellular reservoirs to cells within skin. (1) Chronic activation of NF- κ B in the skin induces expression of adhesion molecules on the surfaces of endothelial cells, increased vascular permeability, and secretion of inflammatory cytokines and chemokines by melanocytes and other resident cells of the skin, (2) recruitment of immune cells and other KSHV-infected monocytes to the vascular endothelium, and (3) extravasation of infected cells from the blood vessels into the underlying dermis, where virus may be transmitted to vascular endothelial cells through cell-to-cell contact or other mechanisms. (4) Accumulation of cell-free and KSHV-infected cells at the superficial dermis, where they interact with and infect resident melanocytes and keratinocytes in the basal membrane.

Article Snippet: Unless otherwise indicated, all rabbit monoclonal antibodies against key components of the NF- κ B pathway, as well as a polyclonal rabbit antibody to phosphorylated NF- κ B p65, were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Transmission Assay, Infection, Activation Assay, Expressing, Permeability

Journal: International Journal of Biological Sciences

Article Title: Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

doi: 10.7150/ijbs.10905

Figure Lengend Snippet: Poly(I:C) induces the phosphorylation of NF-κB and ERK1/2 in human AVICs via TLR3. A. AVICs were treated with 2.5 μg/mL of poly(I:C) for 30 minutes to 24 hours. Representative immunoblots and densitometric data show that treatment with poly(I:C) increases the levels of phospho-NF-κB and phospho-ERK1/2 at 1 hour and 30 minutes, respectively. Phospho-NF-κB is detectable at 4 hours, and phosphorylation levels of ERK1/2 remain elevated at 8 hours in AVICs after being exposed to poly(I:C). B. AVICs were treated with TLR3 siRNA (150 nmol/L; scrambled siRNA as control) 72 hours prior to poly(I:C) stimulation for 2 hours. Representative immunoblots and densitometric data show that treatment with TLR3 siRNA markedly reduces the levels of phospho-NF-κB and phospho-ERK1/2 in cells stimulated with poly(I:C). Data are presented as mean ± standard error of 6 experiments using different AVIC isolates; * P <0.05 vs. untreated control (Ctrl); # P <0.05 vs. poly(I:C) alone; & P <0.05 vs. poly(I:C)+scrambled siRNA.

Article Snippet: Antibodies against phosphorylated nuclear factor-kappa B (NF-κB), total NF-κB, phosphorylated extracellular-regulated kinase1/2 (ERK1/2) and total ERK1/2 were purchase from Cell Signaling, Inc (Beverly, MA).

Techniques: Western Blot

Journal: International Journal of Biological Sciences

Article Title: Activation of TLR3 Induces Osteogenic Responses in Human Aortic Valve Interstitial Cells through the NF-κB and ERK1/2 Pathways

doi: 10.7150/ijbs.10905

Figure Lengend Snippet: Both NF-κB and ERK1/2 are required for the up-regulation of the production of BMP-2, TGF-β1 and ALP by poly(I:C). AVICs were treated with IκB kinase inhibitor (Bay11-7082, 2.5 μmol/L) or a specific ERK1/2 inhibitor (328000ERK, 40 μmol/L) 1 hour prior to poly(I:C) stimulation for 24 hours. Representative immunoblots and densitometric data show that inhibition of either NF-κB ( A ) or ERK1/2 ( B ) abrogates the effect of poly(I:C) on BMP-2, TGF-β1 and ALP production. Data are presented as mean ± standard error of 6 experiments using different AVIC isolates; * P <0.05 vs. untreated control (Ctrl); # P <0.05 vs. poly(I:C) alone or poly(I:C)+DMSO.

Article Snippet: Antibodies against phosphorylated nuclear factor-kappa B (NF-κB), total NF-κB, phosphorylated extracellular-regulated kinase1/2 (ERK1/2) and total ERK1/2 were purchase from Cell Signaling, Inc (Beverly, MA).

Techniques: Western Blot, Inhibition

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: hucMSC Conditioned Medium Ameliorate Lipopolysaccharide-Induced Acute Lung Injury by Suppressing Oxidative Stress and Inflammation via Nrf2/NF- κ B Signaling Pathway

doi: 10.1155/2021/6653681

Figure Lengend Snippet: Effects of CM on LPS-induced Nrf2/NF- κ B signaling pathway changes in lung tissues. Lung tissues were collected at 12 h after LPS challenge for immunostaining and western blot. (a) Immunohistochemistry of phosphorylated NF- κ B p65. (b) Immunofluorescence of Nrf2. (c) Western blot analysis of p65 and p-p65. (d) Western blot analysis of Nrf2. Values presented as mean ± SD. n = 10 mice per group. ∗ P < 0.05 versus the LPS+PBS group; # P < 0.05 versus the control and PBS groups.

Article Snippet: Afterwards, blocking of membranes was done with the 5% skimmed milk for one hour and inoculated overnight with primary antibodies against NF- κ B p65 antibodies (1 : 1000, Cell signaling Technology, United States), phosphorylated NF- κ B p65 antibodies (1 : 1000, Cell signaling Technology, United States), Nrf2 antibodies (1 : 1000, Cell signaling Technology, United States), and Histone H3 (1 : 1000, Cell signaling Technology, United States) in TBST (Tris-buffered saline/Tween) at 4°C.

Techniques: Immunostaining, Western Blot, Immunohistochemistry, Immunofluorescence

Journal: Analytical Cellular Pathology (Amsterdam)

Article Title: hucMSC Conditioned Medium Ameliorate Lipopolysaccharide-Induced Acute Lung Injury by Suppressing Oxidative Stress and Inflammation via Nrf2/NF- κ B Signaling Pathway

doi: 10.1155/2021/6653681

Figure Lengend Snippet: Effects of CM on target genes of the Nrf2/NF- κ B signaling pathway in lung tissues. Lung tissues were collected at 12 h after LPS challenge for real-time quantitative PCR analysis of (a) NQ01, (b) HO-1, (c) GCLC, (d) TNF- α , (e) IL-1 β , and (f) IL-6. Values presented as mean ± SD. n = 10 mice per group. ∗ P < 0.05 versus the LPS+PBS group; # P < 0.05 versus the control and PBS groups.

Article Snippet: Afterwards, blocking of membranes was done with the 5% skimmed milk for one hour and inoculated overnight with primary antibodies against NF- κ B p65 antibodies (1 : 1000, Cell signaling Technology, United States), phosphorylated NF- κ B p65 antibodies (1 : 1000, Cell signaling Technology, United States), Nrf2 antibodies (1 : 1000, Cell signaling Technology, United States), and Histone H3 (1 : 1000, Cell signaling Technology, United States) in TBST (Tris-buffered saline/Tween) at 4°C.

Techniques: Real-time Polymerase Chain Reaction

Journal: BioMed Research International

Article Title: Electroacupuncture Prevents Osteoarthritis of High-Fat Diet-Induced Obese Rats

doi: 10.1155/2020/9380965

Figure Lengend Snippet: Electroacupuncture attenuated systemic and knee inflammation of obese rats. (a) Serum inflammatory markers. (b) Synovial fluid inflammatory markers. (c) Serum and synovial fluid LPS level. (d) Western blot analysis of TLR4, NF- κ B p65, and phosphorylated p65 (P-p65) expression in arthrodial cartilage. All data are expressed as the mean ± SD ( n = 6 per group). Post hoc ANOVA was used to test for statistical significance. ∗ p < 0.05, ∗∗ p < 0.01, and ∗∗∗ p < 0.001 versus the control group; # p < 0.05, ## p < 0.01, and ### p < 0.001 versus the DIO-KOA group.

Article Snippet: After being denatured in boiling for 5 min in SDS sample buffer, 40 μ g of total protein was separated by 6%-15% SDS-PAGE, blotted onto PVDF membranes, and then probed with the following antibodies (Cell Signaling Technology, Danvers, MA, USA): monoclonal anti-TLR4 antibody, monoclonal anti-NF- κ B p65 antibody, monoclonal anti-NF- κ B phosphorylated p-65 (P-p65) antibody, and mouse anti- β -action antibody, conjugated to horseradish peroxidase, which were used as secondary antibodies.

Techniques: Western Blot, Expressing

Journal: Journal of Diabetes Research

Article Title: Novel Mechanisms Modulating Palmitate-Induced Inflammatory Factors in Hypertrophied 3T3-L1 Adipocytes by AMPK

doi: 10.1155/2018/9256482

Figure Lengend Snippet: Effects of AICAR on palmitate-induced MCP-1, NF- κ B, and MAPK signaling in 3T3-L1 adipocytes. 3T3-L1 cells were exposed to 0.3 mmol/L palmitate (black bar) or ethanol vehicle alone (white bar) in the presence or absence of 0.5 mmol/L AICAR for 12 h. The mRNA levels of MCP-1 were measured by quantitative real-time RT-PCR (a). After additional 12 h, cell lysates were immunoblotted to determine the intracellular MCP-1 concentration (b), phosphorylation of NF- κ B on Ser536 (d), ERK1/2 on Thr180/Tyr182 (e), p38 MAPK on Thr180/Tyr182 (f), and JNK on Thr183/Tyr185 (g). β -Actin was measured as an internal control, and each phosphorylation was normalized by the corresponding total protein concentration. The release of MCP-1 was also assessed by ELISA (c). Data are means ± SEM ( n = 4). ∗ p < 0.05, ∗∗ p < 0.01 compared to corresponding control cells. NS: no significant difference compared to corresponding control cells.

Article Snippet: Antibodies against AMPK α , phosphorylated AMPK α , p38 mitogen-activated protein kinase (MAPK), phosphorylated p38 MAPK, phosphorylated p44/42 MAPK (ERK1/2) (Thr202/Tyr204), phosphorylated acetyl-CoA carboxylase (ACC) (Ser79), ACC, NF- κ B p65, phosphorylated NF- κ B p65 (Ser536), phosphorylated JNK, and JNK were all obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Quantitative RT-PCR, Concentration Assay, Protein Concentration, Enzyme-linked Immunosorbent Assay

Journal: Journal of Diabetes Research

Article Title: Novel Mechanisms Modulating Palmitate-Induced Inflammatory Factors in Hypertrophied 3T3-L1 Adipocytes by AMPK

doi: 10.1155/2018/9256482

Figure Lengend Snippet: Effects of A769662 on MCP-1, NF- κ B, and MAPK signaling and intracellular TG contents in 24 h palmitate-preloaded 3T3-L1 adipocytes. Differentiated 3T3-L1 adipocytes were exposed to 0.3 mmol/L palmitate (black bar) or ethanol vehicle alone (white bar) in the presence or absence of 25 μ mol/L A769662 for 24 h. Lysates were immunoblotted to assess the phosphorylation of AMPK on Thr172 (a), NF- κ B on Ser536 (e), ERK1/2 on Thr180/Tyr182 (f), ACC on Ser79 (h), and intracellular MCP-1 (c). The release of MCP-1 was also assessed by ELISA (d). The cellular TG (g) contents were measured and then adjusted to intracellular total protein contents. The levels of MCP-1 mRNA (b) were measured by quantitative real-time RT-PCR at 12 h after stimulation and then normalized over the 18S rRNA signal. Data are means ± SEM ( n = 4). ∗ p < 0.05, ∗∗ p < 0.01 compared to corresponding control cells.

Article Snippet: Antibodies against AMPK α , phosphorylated AMPK α , p38 mitogen-activated protein kinase (MAPK), phosphorylated p38 MAPK, phosphorylated p44/42 MAPK (ERK1/2) (Thr202/Tyr204), phosphorylated acetyl-CoA carboxylase (ACC) (Ser79), ACC, NF- κ B p65, phosphorylated NF- κ B p65 (Ser536), phosphorylated JNK, and JNK were all obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR

Journal: Journal of Diabetes Research

Article Title: Novel Mechanisms Modulating Palmitate-Induced Inflammatory Factors in Hypertrophied 3T3-L1 Adipocytes by AMPK

doi: 10.1155/2018/9256482

Figure Lengend Snippet: Effects of compound C and U126 on MCP-1 in palmitate-preloaded 3T3-L1 adipocytes treated with AICAR. Adipocytes were pretreated with 10 μ mol/L compound C (a, b, c, and d), 10 μ mol/L U126 (e, f), or vehicle (dimethyl sulfoxide) alone for 20 min. Then, the cells were treated with 0.3 mmol/L palmitate (black bar) or vehicle (ethanol) alone (white bar) for 24 h with or without 0.5 mmol/L AICAR. Intracellular MCP-1 (d, f) was quantified by immunoblotting. AMPK phosphorylation on Thr172 (a), ACC phosphorylation on Ser79 (b), NF- κ B phosphorylation on Ser536 (c), and ERK1/2 phosphorylation on Thr180/Tyr182 (e) were also quantified by immunoblot analysis. Each phosphorylation was normalized by the level of the corresponding total protein. β -actin was assessed as an internal control. Results are means ± SEM ( n = 3). ∗ p < 0.05, ∗∗ p < 0.01 compared to the corresponding controls. NS: no significant difference compared to corresponding control cells.

Article Snippet: Antibodies against AMPK α , phosphorylated AMPK α , p38 mitogen-activated protein kinase (MAPK), phosphorylated p38 MAPK, phosphorylated p44/42 MAPK (ERK1/2) (Thr202/Tyr204), phosphorylated acetyl-CoA carboxylase (ACC) (Ser79), ACC, NF- κ B p65, phosphorylated NF- κ B p65 (Ser536), phosphorylated JNK, and JNK were all obtained from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Western Blot

Journal: Clinical and Developmental Immunology

Article Title: Propofol Reduces Lipopolysaccharide-Induced, NADPH Oxidase (NOX 2 ) Mediated TNF- α and IL-6 Production in Macrophages

doi: 10.1155/2013/325481

Figure Lengend Snippet: Effect of propofol pretreatment on LPS-induced phosphorylation of nuclear NF- κ B and Akt. (a), (c) The cells were pretreated with dimethyl sulfoxide (DMSO) or propofol for 40 min and then stimulated with LPS for 1 h. Nuclear NF- κ B and Akt activation, indicated by phosphorylation, was analyzed by Western blotting on whole cell lysates. (b), (d) The levels of phosphorylated NF- κ B (p-NF- κ B) and phosphorylated Akt (p-Akt) were quantified by measuring band intensities and represented as fold change over total NF- κ B and Akt. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P < 0.05) between propofol and LPS treated and LPS only treated groups, respectively.

Article Snippet: The primary antibodies were as follows: NF- κ B (1 : 1000, Cell Signaling Biotechnology), phosphorylated NF- κ B (Ser536) (1 : 500, Cell Signaling Biotechnology), Akt (1 : 1000, Santa Cruz Biotechnology), phosphorylated Akt (Ser 473) (1 : 500, Santa Cruz Biotechnology), and β -actin (1 : 2000, Cell Signaling Biotechnology).

Techniques: Activation Assay, Western Blot

Journal: Clinical and Developmental Immunology

Article Title: Propofol Reduces Lipopolysaccharide-Induced, NADPH Oxidase (NOX 2 ) Mediated TNF- α and IL-6 Production in Macrophages

doi: 10.1155/2013/325481

Figure Lengend Snippet: Effect of NF- κ B Inhibitor on LPS-induced TNF- α and IL-6 expression. RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 20 μ M pyrrolidine dithiocarbamate (PDTC) for 1 h and stimulated with 100 ng/mL LPS for 8 h. The concentrations of TNF- α and IL-6 in culture supernatants were measured by ELISA. (c) RAW264.7 macrophages were pretreated with dimethyl sulfoxide (DMSO) or 20 μ M PDTC for 1 h and then stimulated with LPS for 2 h. Steady state mRNA levels of TNF- α and IL-6 were examined by RT-PCR. (d) The levels of TNF- α and IL-6 mRNA were quantified by measuring band intensities and shown as fold increase relative to β -actin mRNA levels. Each value represents the means ± SD for n = 4. # and ∗ indicate statistically significant differences ( P < 0.05) between propofol and LPS treated and LPS only treated groups, respectively.

Article Snippet: The primary antibodies were as follows: NF- κ B (1 : 1000, Cell Signaling Biotechnology), phosphorylated NF- κ B (Ser536) (1 : 500, Cell Signaling Biotechnology), Akt (1 : 1000, Santa Cruz Biotechnology), phosphorylated Akt (Ser 473) (1 : 500, Santa Cruz Biotechnology), and β -actin (1 : 2000, Cell Signaling Biotechnology).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction